ABSTRACT
Although the guiding principles for molecular identification of traditional Chinese medicines (TCM) using DNA barcoding have been recorded in the Chinese Pharmacopoeia, there is still a lack of systematic research on its application to commercial TCM decoctions. In this study, a total of 212 commercial TCM decoctions derived from different medicinal parts such as root and rhizome, fruit and seed, herb, flower, leaf, cortex, and caulis were collected to verify applicability and accuracy of the method. DNA barcodes were successfully obtained from 75.9% (161/212) of the samples, while other samples failed to be amplified due to genomic DNA degradation. Among the 161 samples, 85.7% of them were identified as recorded species in the Chinese Pharmacopoeia (2020 edition). In addition, 14 samples could be identified as species recorded in the Chinese Pharmacopoeia and their closely related species in the same genus. Morphological identification for the unconfirmed samples showed that eight were genuine species and three were adulterants, while the other three were unidentifiable due to lack of morphological characteristics. Furthermore, the DNA barcodes of seven samples accurately mapped to the sequences of adulterants. Remarkably, counterfeit products were detected in two samples. These results demonstrate that DNA barcoding is suitable for the identification of commercial TCM decoctions. The method can effectively detect adulterants and is appropriate for use throughout the industrial chain of TCM production and distribution, and by the supervisory agencies as well.
ABSTRACT
In order to explore the use of DNA barcode in the identification of wild Phytolacca resources in the Shaanxi Guanzhong area, 29 DNA samples were amplified and sequenced by using the universal primers ITS2 and psbA-trnH. The sequences were spliced and proof-read by Codon CodeA aligner V3.0, followed by blast comparison and identification analysis; mega 6.0 was used to analyze sequence characteristics, Kimura 2-Parameter (K2P) was used to analyze distance and intraspecific or interspecific variation, and Neighbor-Joining trees were established to evaluate the ability of two pairs of candidate sequences to distinguish Phytolaccae Radix from its adulterants. The results showed that the success rate of PCR amplification and sequencing of ITS2 and psbA-trnH was 100%; the NJ tree showed that both ITS2 and psbA-trnH sequences could separate P. acinosa, P. americana, other species of the same genus like P. japonica, P. exiensis and two adulterant species into a single clade; primer ITS2 had an advantage over psbA-trnH in determining interspecific genetic distances. Therefore, both ITS2 and psbA-trnH sequences can be used for identification of Phytolacca and their adulterants, which provides a theoretical basis for the distribution of wild Phytolacca resources and their rational development and utilization.
ABSTRACT
Based on the ITS2 and psbA-trnHsequences, molecular biological identification and genetic relationship of Fritillaria cirrhosa with its relative species were carried out. In this paper, the PCR-RFLP method specified by the Chinese Pharmacopoeia was performed on all samples at first. Secondly, the ITS2 and psbA-trnH sequences of all samples were amplified. Then, the amplified products were used to analyze the genetic distance, construct the phylogenetic tree, assess the identification efficiency, and evaluate the genetic relationship as well. The result showed that all the samples were divided into two groups by PCR-RFLP method. The samples in the first group, including Fritillaria ussuriensis, Fritillaria thunbergii and Fritillaria pallidiflora, could not be digested by SmaI, while the other samples in the second group, including Fritillaria mellea, Fritillaria sinica, Fritillaria cirrhosa var. ecirrhosa Franch, Fritillaria unibracteata var. longinectarea and Fritillaria cirrhosa, could be digested by SmaI. Then, ITS2 and psbA-trnH sequences of all samples were obtained. The length of various ITS2 sequences were distributed from 235 to 239 bp, and the average intra- and inter-specific genetic distance were 0.001 and 0.022, respectively. NJ tree showed that all samples were separated into "Northern Fritillaria" group (Fritillaria ussuriensis and Fritillaria pallidiflora) and "Southern Fritillaria" group (Fritillaria thunbergii, Fritillaria mellea, Fritillaria sinica, Fritillaria cirrhosa var. ecirrhosa Franch, Fritillaria unibracteata var. longinectarea and Fritillaria cirrhosa). The latter group could be further divided into Fritillaria thunbergii and Fritillaria cirrhosa subgroup, and the species in Fritillaria cirrhosa subgroup had close phylogenetic relationships. The length of psbA-trnH sequences was distributed from 337 to 373 bp, and the intra- and inter-specific genetic distance were 0.263 and 0.329, respectively. The samples in this paper could not be clustered effectively by NJ tree. This indicated that the ITS2 sequences were not only able to identify Fritillaria cirrhosa with its partial relative species quickly and accurately, but also clarify the relationship between different Fritillaria species. Therefore, it provided an important theoretical foundation for the development of molecular markers, effective protection, and rational development and utilization of Fritillaria resources.